Cell therapy has emerged as a potential new treatment to reduce injury and improve outcome after ischemic stroke. Several studies have demonstrated that mononuclear cells (MNCs) from bone marrow are safe and enhance recovery in animal stroke models. This grant proposal aims to define specific mechanisms by which MNCs reduce neurological deficits using a variety of in vitro and in vivo models of ischemic stroke. Our preliminary data indicate that MNCs are cytoprotective and reduce pro-inflammatory responses in the post-ischemic brain. In Specific Aim 1, we first will determine which cell populations within MNCs are critical to reduce neurological deficits, lesion size, and pro-inflammatory responses after stroke. In Specific Aim 2, we will test the hypothesis that MNCs release the cytokines, IL-10 and IGF-1, which lead to neuroprotection and modulation of microglia. Aim 2A will focus on the hypothesis that both cytokines, secreted by MNCs, directly protect neurons in in vitro models of hypoxia and directly reduce injury in an animal model of stroke. Aim 2B addresses the hypothesis that MNCs activate and change microglia to become anti-inflammatory and neuroprotective after stroke. We hypothesize that IL-10 and IGF-1 are key factors in the capacity of MNCs to modulate the anti-inflammatory and neuroprotective effects of microglia after stroke. Our proposed studies would be the first step to elucidate specific pathways mediating the protective effects of MNCs in the post-ischemic brain and are critical to develop MNCs as a potential treatment for ischemic stroke, a condition for which there is an enormous public health need for more and improved therapies.